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Quantification of the glycogen 13 C‐1 NMR signal during glycogen synthesis in perfused rat liver
Author(s) -
Bergans N.,
Dresselaers T.,
Vanhamme L.,
Van Hecke P.,
Van Huffel S.,
Vanstapel F.
Publication year - 2003
Publication title -
nmr in biomedicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.278
H-Index - 114
eISSN - 1099-1492
pISSN - 0952-3480
DOI - 10.1002/nbm.812
Subject(s) - glycogen , glycogenolysis , glycogen branching enzyme , nuclear magnetic resonance spectroscopy , glycogenesis , glycogen synthase , chemistry , endocrinology , medicine , biochemistry , biology , stereochemistry
Abstract We studied glycogen synthesis from glucose in perfused livers of fed ( n = 4) and 24 h starved ( n = 7) rats. Glycogenolysis was inhibited by BAY R3401 (150 µ M ) and proglycosyn (100 µ M ). After 60 min, we replaced 99% 13 C‐1 glucose by natural abundance glucose. This pulse‐chase design allowed us to recognize residual ongoing futile glycogen turnover from the release of initially deposited 13 C‐label, into the 13 C‐free chase medium. Net residual turnover was less than 2 ± 0.7% and 0.6 ± 0.2% of 1‐ 13 C glycogen deposition rates of 0.31 ± 0.04 and 0.99 ± 0.04 µmol glucose g −1 min −1 , in starved and fed livers, respectively. The 1‐ 13 C glycogen signal was monitored throughout the experiment with proton‐decoupled 13 C NMR spectroscopy and analyzed in the time domain using AMARES. We noticed progressive line‐broadening in any single experiment in the chase phase. One or a sum of two to three overlapping Lorentzians, with different exponential damping factors, were fitted to the signal. When the S/N was better than 40, the fit always delivered a small and a broad component. In the chase phase, the fit with a single Lorentzian resulted in a decline of glycogen signal by about 15 ± 4 and 12 ± 2% in starved and fed rats, respectively. This apparent decline in 1‐ 13 C glycogen signal could not be accounted for by the appearance of equivalent amounts of 13 C‐labeled metabolites in the perfusate. The fit with a sum of two Lorentzians resulted in a decline of glycogen signal intensity of 7 ± 5 and 5 ± 3% in starved and fed rats, respectively, which reduced the apparent turnover to 8 ± 9% and 6 ± 4%, respectively. Quantification of the growing 13 C‐1 glycogen signal requires a model function that accommodates changes in line shape throughout the period under study. Copyright © 2003 John Wiley & Sons, Ltd.